A defined basal medium free of complex biological ingredients has been developed for axenic cultivation of Entamoeba histolytica. Based on their zymodemes (isoenzyme profiles), believed to be a stable and inherent property, isolates of E. histolytica have been divided into two groups, nonpathogenic and pathogenic. Todate, only members of the latter have been axenized. Axenization of cloned isolates of amebae displaying nonpathogenic zymodemes from healthy humans is reported. Furthermore, these zymodemes can be altered by manipulating the bacterial flora associated with the amebae in vitro. Axenization of amebae displaying a nonpathogenic profile resulted in a shift to a pathogenic type. Reassociation of the amebae with their bacterial flora resulted in return to the original zymodeme. Reassociation of axenized amebae, isolated from patients with amebic disease and displaying pathogenic zymodemes, with bacterial flora from healthy carriers resulted in shifts from pathogenic to nonpathogenic zymodemes. Reaxenization restored the profiles to their original state. Changes in virulence always accompanied shifts in zymodeme. Amebae displaying a pathogenic zymodeme were virulent, and avirulent when displaying a nonpathogenic zymodeme. Three classes of molecular probes with diagnostic potential were developed for E. histolytica. The first consists of protein profiles of E. histolytica and related Entamoeba made by mapping biosynthetically radio-labeled proteins separated by 2-dimensional gel electrophoresis, the second is a catalog of species-specific monoclonal antibodies, and the third consists of isolate-and-species-specific repeated DNA fragments cloned in plasmid vector pTz18R.